Liver directed gene therapy with recombinant AAV vectors has had recent success in human patients with hemophilia B. However, several problems remain to be solved before gene therapy can be broadly applied to the majority of inborn errors affecting the liver. First, hepatocytes turn over - albeit slowly - in both humans an rodents even without injury. Since rAAV remains mostly episomal and is lost with cell replication it is predictable that transgenes introduced by current generation rAAVs will not persist indefinitely. Second, the limited size capacity of AAV makes it poorly suited for disorders caused by mutations in large genes - for example hemophilia A. Third, the vector doses required to achieve efficient in vivo transduction in human are high and lower doses would be desirable from a manufacturing/cost as well as safety perspective. Finally, the currently used minigene cDNA expression cassettes result in unregulated and non-physiologic levels of transgene expression, which may prove to be problematic for the treatment of some disorders. Here we propose to further develop 3 distinct approaches to enhance transgene persistence in liver gene therapy. In aim 1, we will work on gene rAAV mediated gene repair in hepatocytes. Aim 2 is focused on rAAV vectors that can integrate into ribosomal DNA as a safe harbor. In aim 3 we will refine the components of a system designed to achieve in vivo selection of genetically modified hepatocytes using a small molecule inhibitor of fumarylacetoacetate hydrolase (Fah).